By adapting approaches that have been applied with great success to testing the sea urchin developmental gene regulatory network, we propose to perform a detailed analysis of the gene interactions involved in specifying vertebrate neural crest cells. Our aim is to understand the genomic control of this process at a systems level by revealing most/all of the inputs into the system and methodically functionally perturbing them to examine interactions amongst players. In other words, we propose to test a putative neural crest gene regulatory network (NC-GRN) at a systems levels in a single vertebrate. These efforts will be greatly facilitated by the advent of new, high speed technologies that will significantly increase the rate of data acquisition and interpretation as well as novel bioinformatics tools to interrogate genomic information. We will draw heavily on methodologies and concepts developed in the Davidson lab. The goal is to apply these to a vertebrate system at moderate to high throughput. This represents a huge leap forward in both the scale and depth of what can be tested. The recent availability of the chick genome affords a rich tool for discovery of genes and regulatory regions. In addition as an amniote, chick development is similar to humans and, unlike mammals, is accessible to imaging at early stages since the embryo develops outside the mother. We will test linkages in the chick neural crest gene regulatory network, identify regulatory elements and test direct interactions. Aim 1: Examine effects of loss-of-function of known neural plate border and neural crest specifiers. By introducing morpholino antisense oligonucleotides into the prospective neural plate border or closing neural tube. Effects on potential downstream targets will be examined by in situ hybridization and QPCR. Aim 2: Test the function of newly identified transcription factors in the NC-GRN We will test the role and position additional transcription factors in the network and we will continue to attempt to identify transcription factors that feed into the NC-GRN. Aim 3: Isolate regulatory regions of neural crest specifer and downstream targets. We will isolate putative regulatory regions of neural crest specifier genes, initially for Sox 10 and then other specifiers, via comparative sequence analysis. Candidate regions will be electroporated into early chick embryos to identify neural crest regulatory elements. Aim 4: Establish direct relationships within the network by identification of transcription factor binding sites within regulatory regions of downstream genes. We will interrogate the regulatory regions of neural crest enhancer elements for critical sequences responsible for binding of neural plate border specifiers genes and/or neural crest specifier genes. We will assay for direct binding interactions within the network using chromatin immunoprecipitation assay, electrophoretic mobility shift assays and mutational analysis.